Nano-Bio-Technology and Sensing Chips: New Systems for Detection in Personalized Therapies and Cell Biology

Further advances in molecular medicine and cell biology also require new electrochemical systems to detect disease biomarkers and therapeutic compounds. Microelectronic technology offers powerful circuits and systems to develop innovative and miniaturized biochips for sensing at the molecular level. However, microelectronic biochips proposed in the literature often do not show the right specificity, sensitivity, and reliability required by biomedical applications. Nanotechnology offers new materials and solutions to improve the surface properties of sensing probes. The aim of the present paper is to review the most recent progress in Nano-Bio-Technology in the area of the development of new electrochemical systems for molecular detection in personalized therapy and cell culture monitoring

Label-free detection of tuberculosis DNA with capacitive field-effect biosensors

The detection of pathogens from sample material from infected patients is the basis on which a considerable medical diagnosis can be made. Pathogens can be clearly identified based on their genomic material (DNA). A large number of different DNA detection methods with individual advantages and disadvantages have been established. If such methods should be used for certain applications, e.g. for point-of-care measurements, there are a number of requirements which should be considered: A measurement must be performed very fast, inexpensive, simple and reliable. It has been shown that label-free detection principles in particular the field-effect based detection-methods, meet the given requirements. In this thesis, the development of a new measuring method for the detection of DNA (with sequences from Mycobacterium tuberculosis) using a field-effect sensor, is described. The electrolyte-insulator-semiconductor (EIS) structure was selected as the basis for the sensor chip because it has the simplest structure of all field-effect sensors and is inexpensive to manufacture. EIS sensors are capacitive structures that can be read out using an impedance analyzer. The measured value is directly related to the surface potential of the sensor. If the DNA, which is negatively charged in solution, is brought close to the sensor surface, this causes a change in the surface potential via a change in the charge situation on the chip surface. This change in potential can be read out with the help of the EIS sensors: The detection method is based on the detection of a hybridization event on the sensor surface. The surface is modified with a probe single-strand DNA (ssDNA) which has a known sequence that is complementary to the ssDNA that is intended to be detected. As soon as the target ssDNA reaches the surface, a hybridization can occur, whereby a signal shift can be measured. The signal shift is caused by the additional negative charge of the hybridized target DNA molecules. In the case of non-complementary target DNA (ncDNA), there is no hybridization and the signal remains constant. The immobilization of the catcher ssDNA was carried out with by a surface modification using positively-charged polyelectrolyte (poly (allylamine hydrochloride), PAH). Compared to other immobilization strategies that are described in the literature, the capture ssDNA binds adsorptively to the sensor surface, which simplifies the preparation and can be carried out quickly and cheaply. The main topics of this thesis cover the selection of the sensor layout (EIS sensor with SiO2 as surface oxide), the selection and optimization of the surface modification (using PAH), the verification of the forming of double-stranded DNA and the evaluation of the measurement data acquisition by means of capacitive measurements. Due to the adsorptive binding, the DNA strands likely lie flat on the sensor surface. This means that the negative charge of the DNA is located closely to the surface, which means that a high measurement signal can be recorded. The developed protocol was also used with light-addressable potentiometric sensors (LAPS). LAPS are structurally very similar to EIS sensors and have the advantage that they can detect changes in the surface potential in a spatially resolved manner. This makes it possible, for example, to arrange an array so that several DNA experiments can be carried out simultaneously on one chip. However, the measurement setup is more complex because of the necessity of a light source. The measurement of the DNA hybridization on the sensor surface was realized by using the developed method: PAH/ssDNA-modified EIS chips were brought into contact with cDNA solutions. Measurable surface potential changes could show that the hybridization was successful. In direct comparison with experiments where ncDNA was applied to the modified sensor, signal differences of about 11 times higher were measured for cDNA than for ncDNA. The developed method also allows a very simple reuse of the chip by just a repeating of the modification steps on an already used chip. This reusability of the sensors was investigated by performing up to five repetitive surface modification and DNA attachment experiments sequentially with just one chip. A steady decrease in the sensor signal could be observed after each additional layer (PAH or DNA); however, this observation is related to the Debye screening effect. Finally, the developed biosensor was used to detect PCR-amplified cDNA. A detection of the target cDNA was successful and significant, although the additional (interfering) substances in the solution, that were necessary for the PCR process (enzymes, etc.), disturbed the measurement signal. Measurements in which a concentration series of cDNA were used to determine the lower detection limit (0.3 nM) and the sensitivity (7.2 mV / decade). Extracted and amplified target DNA from Mycobacterium tuberculosis-spiked human saliva-samples was also examined using the method. A clear differentiation between positive and negative material could be recognized with the help of the PAH / ssDNA-modified EIS sensor chips. All developed process steps were validated using fluorescence measurements as a reference method. With the PAH-modified capacitive field-effect biosensor, that was developed in this thesis, a quick, simple and inexpensive measurement platform for the DNA hybridization reaction is given. The detection of amplified genomic DNA from real Mycobacterium tuberculosis-spiked saliva samples underlines the potential of this procedure as a sensor approach for pathogen detection for medical applications

Developing nucleic acid-based electrical detection systems

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed

Advanced Electrochemical Biosensors

With the progress of nanoscience and biotechnology, advanced electrochemical biosensors have been widely investigated for various application fields. Such electrochemical sensors are well suited to miniaturization and integration for portable devices and parallel processing chips. Therefore, advanced electrochemical biosensors can open a new era in health care, drug discovery, and environmental monitoring. This Special Issue serves the need to promote exploratory research and development on emerging electrochemical biosensor technologies while aiming to reflect on the current state of research in this emerging field

Dual parametric sensors for highly sensitive nucleic acid detection

The primary focus of this research work was on the design and development of a molecular scale (nano-scale) capacitive sensing mechanism for the highly sensitive and label-free detection of Nucleic Acid hybridization. These novel capacitive sensors with nano-scale electrode spacing offer solutions to many problems suffered by the conventional signal transduction mechanisms, thereby immensely improving the sensitivity of the biomolecular detection processes. Reducing the separation between the capacitive electrodes to the same scale as the Debye length of the sample solution, results in the overlapping of the electrical double layers of the two electrodes, thereby confining them to occupy a major fraction of the dielectric volume. This decreases the potential drop across the electrodes and thus dielectric measurements at low frequencies are made possible. The dielectric properties during hybridization reaction were measured using 10- mer nucleotide sequences. A 30-40% change in relative permittivity (capacitance) was observed due to DNA hybridization at 10Hz, which is much more sensitive than the previously reposted detection measurements (2-8% signal change). In parallel to the above work, a second label-free sensing mechanism based on field effect capacitive sensors with Metal-Oxide-Semiconductor (MOS) structure has been developed and its ability to provide real-time monitoring of oligonucleotide immobilization and hybridization events are studied. The immobilization of probe oligomers on the sensor surface and their hybridization with the target oligomers of complimentary sequences has produced significant shifts (140mV and 73mV respectively) in the Capacitance-Voltage characteristics measured across the device. In an attempt to utilize the individual merits of the nano-scale electrochemical capacitive sensor and the field effect MOS capacitive structure, a novel dual parametric sensing architecture comprising of both these transducing elements on a single sensor is designed. The detection scheme based on the combined analysis of the two parameters- Dielectric property and intrinsic molecular charge- of Nucleic acid molecules has found to reveal complimentary information of significance about the analyte-probe interactions. As a separate experiment the applications and promises of a novel technique of enhancing the speed and selectivity of the molecular detection processes by the application of an external electric field of precisely controlled intensity was studied. Experiments were conducted with 10-mer sequences and proved the feasibility of this technique in inducing in providing a faster and selective immobilization and hybridization reactions. The research work in this direction has been in collaboration with the Rational Affinity Devices, LLC, a New Jersey based corporation. The above mentioned biosensing mechanisms and detection techniques have the advantage of simplifying the readout and increasing the speed and ease of nucleic acid assays, which is especially desirable for characterizing infectious agents, scoring sequence polymorphism and genotypes, and measuring mRNA or miRNA levels during expression profiling. Once fully optimized and well assembled they have great potential to be developed in to a commercial full-scale biosensor capable of providing high-value diagnostic testing at the point of patient care places

New insights for using self-assembly materials to improve the detection stability in label-free DNA-chip and immuno-sensors

This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene-glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene-glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8-11 nF/mm(2) for antibody layers and close to 6 nF/mm(2) for DNA probes. It is in the range of 10-12 nF/mm(2) and of 4-6 nF/mm(2) for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11-37% and of 0,23-0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene-glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene-glycol functional groups and to solution ions not entering into probe films is finally proposed. (C) 2008 Elsevier B.V. All rights reserved

Establishment of surface functionalization methods for spore-based biosensors and implementation into sensor technologies for aseptic food processing

Aseptic processing has become a popular technology to increase the shelf-life of packaged products and to provide non-contaminated goods to the consumers. In 2017, the global aseptic market was evaluated to be about 39.5 billion USD. Many liquid food products, like juice or milk, are delivered to customers every day by employing aseptic filling machines. They can operate around 12,000 ready-packaged products per hour (e.g., Pure-Pak® Aseptic Filling Line E-PS120A). However, they need to be routinely validated to guarantee contamination-free goods. The state-of-the-art methods to validate such machines are by means of microbiological analyses, where bacterial spores are used as test organisms because of their high resistance against several sterilants (e.g., gaseous hydrogen peroxide). The main disadvantage of the aforementioned tests is time: it takes at least 36-48 hours to get the results, i.e., the products cannot be delivered to customers without the validation certificate. Just in this example, in 36 hours, 432,000 products would be on hold for dispatchment; if more machines are evaluated, this number would linearly grow and at the end, the costs (only for waiting for the results) would be considerably high. For this reason, it is very valuable to develop new sensor technologies to overcome this issue. Therefore, the main focus of this thesis is on the further development of a spore-based biosensor; this sensor can determine the viability of spores after being sterilized with hydrogen peroxide. However, the immobilization strategy as well as its implementation on sensing elements and a more detailed investigation regarding its operating principle are missing. In this thesis, an immobilization strategy is developed to withstand harsh conditions (high temperatures, oxidizing environment) for spore-based biosensors applied in aseptic processing. A systematic investigation of the surface functionalization’s effect (e.g., hydroxylation) on sensors (e.g., electrolyte-insulator semiconductor (EIS) chips) is presented. Later on, organosilanes are analyzed for the immobilization of bacterial spores on different sensor surfaces. The electrical properties of the immobilization layer are studied as well as its resistance to a sterilization process with gaseous hydrogen peroxide. In addition, a sensor array consisting of a calorimetric gas sensor and a spore-based biosensor to measure hydrogen peroxide concentrations and the spores’ viability at the same time is proposed to evaluate the efficacy of sterilization processes

A Rapid and Ultra-sensitive Biosensing Platform based on Tunable Dielectrophoresis for Robust POC Applications

With the ongoing pandemic, there have been increasing concerns recently regarding major public health issues such as abuse of organophosphorus compounds, pathogenic bacterial infections, and biosecurity in agricultural production. Biosensors have long been considered a kernel technology for next-generation diagnostic solutions to improve food safety and public health. Significant amounts of effort have been devoted to inventing novel sensing mechanisms, modifying their designs, improving their performance, and extending their application scopes. However, the reliability and selectivity of most biosensors still have much to be desired, which holds back the development and commercialization of biosensors, especially for on-site and point-of-care (POC) usages. Herein, we introduce an innovative two-phase sensing strategy based on tunable AC electrokinetics and capacitive sensing. By dividing the detection process into a sensitivity-priority step and a selectivity-priority step, the specificity and sensitivity of a biosensor can be significantly improved. A capacitive POC aptasensor is fabricated for the implementation of the 2-phase detection and a quasi-single-cell level detection of limit together with an excellent selectivity is achieved simultaneously. The sensor is capable of handling real-world clinic samples without sophisticated pretreatment. Just after a simple one-step dilution, the developed sensor can detect bacteria no less than 2~3 bacteria/10 µL in raw milk samples within 100 s. Alongside whole bacteria detection, the biosensor can also detect endotoxin, the lipopolysaccharide, in bovine serum samples, with a limit of detection of 10 pg/mL. The biosensor is low-cost and easy to use. This work not only demonstrates a biosensor with significant advantages in sensitivity, selectivity and assay time but also opens up a new horizon for further research of all affinity-based biosensors

New Trends in Impedimetric Biosensors for the Detection of Foodborne Pathogenic Bacteria

The development of a rapid, sensitive, specific method for the foodborne pathogenic bacteria detection is of great importance to ensure food safety and security. In recent years impedimetric biosensors which integrate biological recognition technology and impedance have gained widespread application in the field of bacteria detection. This paper presents an overview on the progress and application of impedimetric biosensors for detection of foodborne pathogenic bacteria, particularly the new trends in the past few years, including the new specific bio-recognition elements such as bacteriophage and lectin, the use of nanomaterials and microfluidics techniques. The applications of these new materials or techniques have provided unprecedented opportunities for the development of high-performance impedance bacteria biosensors. The significant developments of impedimetric biosensors for bacteria detection in the last five years have been reviewed according to the classification of with or without specific bio-recognition element. In addition, some microfluidics systems, which were used in the construction of impedimetric biosensors to improve analytical performance, are introduced in this review